Animal feed composition for prevention of mycotoxicoses and method for preparing same



This application is a continuation-in-part of my copending application Serial No. 850,075, filed November 2, 1959, now abandoned, which is a continuation-in-part of my application Serial No. 582,323, filed May 3, 1956, now abandoned. 4

This invention relates to cereal compositions wherein Y the elaboration of fungal metabolic by-products, which are toxic to animalsingesting the same, is inhibited. -Furthermore, the invention is adapted to animal feed com positions containing 2-chloro-5-nitropyridine for the prevention of mycotoxicoses in domestic livestock and other animals.

Poultry hemorrhagic syndrome is an example of a mycotoxicoses and is characterized by certain abnormal clinical symptoms and by hemorrhages in the legs, thighs and other organs, as well as by degenerative changes in the liver and kidneys, and by a pronounced depression of the blood-forming tissues.

Although death losses as a result of the hemorrhagic syndrome range from to over 50 percent of the afliicted birds and morbidity up to 90 percent, the significance of this disease has even greater economic implications. Many chickens which may appear clinically normal are downgraded during dressing, because of hemorrhages in the musculature of the legs, thighs and breast.

In the past, various theories have been advanced as possible causes of this elusive disease, but with no conclusive evidence that any of the advanced theoretical factors has been responsible for the classical field syndrome. For example, one of the most popular theories centers on avitaminosis K induced by sulfaquinoxaline toxicity, or by antagonism of this coccidiostat or other drugs to vitamin K. However, the fact that the syndrome has occurred in the complete absence of sulfaquinoxaline or other drugs regarded as antimetabolitic to vitamin K precludes this theory. The avitaminosis K theory is invalidated further by the fact that very little beneficial response was elicited from the administration of therapeutic quantities of vitamin K to the afflicted birds. Therefore, methods used in the past for the amelioraticin of this hemorrhagic disease have not been successfu I have discovered that toxic elaborated products as the result of growth of fungi on cereals fed to animals are the basic cause of this hemorrhagic disease. Thus, the hemorrhagic syndrome may be classified as being a mycotoxicoses, which is a toxemia produced in animals that have consumed toxic substances secreted by fungi. The relationship of the toxic fungal metabolic products to the hemorrhagic syndrome has been confirmed conclusively by production of the hemorrhagic syndrome under laboratory and field conditions.

Therefore, a principal object of my invention is the prevention of those maladies associated with ingestion of cereals, hereinafter defined, such as straw, hay, grain, and processed feed on which fungi proliferate as saprophytes and give rise to toxic products, either by secretion of metabolic productsinto the substrata, or by the toxic substance being present as an endotoxin in the mycelium or in the fruiting bodies of the fungus. Because of the possibility that small portions of infected cereals may stand for a considerable period of time before they are United States Patent 0 3,208,852 Patented Sept. 28, 1965 ingested, it is necessary that the cereals be protected against development of. toxic fungal metabolic by-products for a long'period of time under adverse conditions.

It is with the prevention of such cereal borne intoxication Once having determined the etiology of the hemorrhagic syndrome and having classified it as a mycotoxicoses, I

then discovered that the production of toxic materials, or

at least toxic levels of these materials, could be suppressed by the addition of 2-chloro-5-nitropyridine to thecereal substrate. I further discovered that this could be accomplished by the addition of 10 to 1000 p.p.m. of

the 2-chloro-5-nitropyridine to the cereal substrate. The preferred concentration being within the range of 30 to 500 p.p.m. V

It has been found that the occurrence of mycotoxicoses can be prevented by the use of Z-chloro-S-nitropyridine in less than fungicidal concentrations. True mechanism of activity is unknown; however, it is believed that small amount of the Z-chIOrO S-nitropyridine interfere with the metabolism of the fungi and thereby prevent the elaboration of toxic by-products. While there may be a proliferation of the fungi in the cereal, a subsequent ingestion of the infected cereal with less than fungicidal concentrations of 2-chloro-5-nitropyridine does not produce manifestations of mycotoxicoses. I

Therefore, the invention relates to a cereal composition, containing a cereal which when infected with fungi is normally capable of producing toxic levels of metabolic by-products in conditions of relatively high humidity, and a concentration of 2-chloro-5-nitropyridine, which is within the range of 10 to 1,000 p.p.m.

For the reasons outlined above, a concentration of 10 to 400 p.p.m. in poultry feed, for example, does not necessarily suppress profuse fungal proliferation in inoculated feed; however, an ingestion of this feed by chicks does not result in any manifestations of the poultry hemorrhagic syndrome. Therefore, although there may be profuse fungal proliferation in the inoculated feed, there is suflicient suppression of the poultry hemorrhagic syndrome.

10 to 1000 p.p.m., in cereals which have been inoculated.

with suspensions of the aforementioned fungi.

The following examples are provided for illustrative purposes and may include particular features of the invention. However, the examples should not be construed as limiting the invention, many variations of which are possible without departing from the spirit or scope thereof.

EXAMPLE l.PREPARATION OF ANIMAL FEED FOR HEMORRHAGIC SYNDROME STUDY Eight kilograms of non-sterile broiler mash were weighed and themoisture content of the feed determined by means of a Delrnhorst Moisture Detector. To the sample were added 40ml. of an aqucoussuspension ,of if a mixture of toxic fungi (Alternaria sp., Aspergilius clovatils, Aspergillus fumigatus, Aspcrgillus fluvus, Aspergilchlorine to bring the total moisture content'ofthe feed to approximately 21 percent. The-fungi had been isolated from feed and litter obtained from broiler'houses in which'the poultry hemorrhagic syndrome was enzootic. 1 The moistened sample was then' premixed by hand, rubbed through a /s inch mesh stainless steel screen and mixed mechanically for 5 minutes in a Hobart feed mixer. The sample was then divided into two lots, and to a small portion or one of the lots a small quantity of'2-.

chloro-S-nitropyridinewas added. After thoroughly premixing the 2-chloro-5nitropyridine with a spatula, more I feed was added from the lot from which the small portion wastaken, and premixed by hand until the whole lot was premixed, and this was continued untilthe -2- chloro-S-nitropyridine content was 500 p.p.m.,, based on the weight of the moist feed. The lot containing the 2-chloro-5-nitropyridine (Lot 2) was then further mixed mechanically for 5 minutes in the Hobart feedmixer.

Lot 1, which contained no- 2-chloro-5-nitropyridine, served as a control. Both sample lots were then transferred to round fiberboard containers, equippedwith a gauze-covered opening in the lid for ventilation, and incubated at ordinary room temperatures. The sample lots were observed periodically for gross and microscopic proliferation of fungi.

After 14 days, a very heavy fungalgrowthand formation offruiting bodies was observed in Lot ,1. After 18 days, there was a heavier fungal growth observed and further formation of fruiting bodies in Lot 1, and no growth was observed in Lot 2. On the th day both sample lots were removed and dried for 48 hours in incubators adjusted to approximately 45 C.

EXAMPLE 2.DETERMINATION OF THE EFFI- CACY OF Z-CHLORO-NITROPYRIDINE IN THE PREVENTION OF MYCOTOXICOSES During the early stages of the poultry hemorrhagic syndrome, the affected birds show signs of depression, diarrhea which is frequently bloody, paleness of combs and Wattles, anorexia and varying morbidity and mortality. Autopsy findings in such birds reveal hemorrhages and congestion in the musculature of the legs, thighs and breast, and in many other tissues, as well as erosion of the mucosa of the proventriculusand gizzard. The other tissue changes are characteristic of an acute degenerative condition in the liver and'kidneys.

Autopsy findings, in 'birds which have progressed to the advanced syndrome, will reveal blood pigments in the liver, proteinaceous and hematogenous castsdn the distal convoluted and collected tubules of the kidneys, and paleness, and gelatinization'of, and depression in, the blood-forming elements inthe bone marrow.

Two groups of New Hampshire-Red Barred Rock scross, day-old chicks, 20 per group, were fed ad libitum, re-

added, showed none of the early symptoms of the hemorrhagic syndrome. The chicks of this group grossly appeared normal for the fullperiod of the test.- f

. Additional lotsof feed were prepared f llowing the procedure setforth he'reinabove'. At the end offour I weeks, .the supply of feed was exhausted, and 'IOchicks from each group were sacrificedby decapitation, and examined grossly at autopsy for manifestations of the syndrome. a

These test results show that there is suppression of the hemorrhagic syndrome. This is shown by the autopsy, as well as 'by' gross observation which revealed a pronounced suppression of the hemorrhagic syndrome. Attempting to achieve gre atenlucidity, as well 'as speed, in the. interpretation of the results of the autopsy, an arbitrary hemorrhagic syndrome score was: assigned to the twelve organs examined at autopsy. A normal organ is given the value zero, slight manifestations 1, moderate manifestations 2, pronounced manifestations 3. The .score is then totaled for each of the,..carcasses examined, and a mean score is determined by dividing the total score ofall the carcasses examined by the number of carcasses. The results of the autopsy of the two groups, of chicks are set forth in. the following table.

EXAMPLE 3.COMPARATIVE,STUDIES OF THE EFFICACY OF LESS THAN FUNGICIDAL CON- CENTRATIONS OF 2 CHLORO 5 NIT ROPYRI- 'DINE, AND FUNGICIDAL CONCENTRATIONS OF 2 CHLORO 5 NITROPYRIDINE' ON THE SUPPRESSION OF THE'HEMORRHAGIC SYN- DROME 1 Nine lots of non-sterile broiler. mash containing no anti- "biotic or coccidiostate were inoculated with a mixture of toxic fungi (Alternaria ,sp., Aspergillus clavatus, Aspergillus. fumigatus, Aspergillus flavus, Aspergillun glaucus, Pdecilomyces varioti, Penicillium' purpurogenum, Penicillum, rubrum, and-three unidentified species of Penicillia),

I adjusted'to 21.8 percent moisture, and containing respectively, the following lots of feed: the inoculum-con spect-ively, 0, 10, 50,100, 200, 250, 300, 400 and 500 p.p.m. of 2-chloro-5-nitropyridine, were incubated attordinary room temperatures, observed for fungal'proliferar tion over a period to 13 to 14 days, then dried and fed to day-old chicks, 10 animals per group. The chicks, which were maintained on batteries, had free access to water and the prepared feeds, and were observed daily for gross manifestation of toxicity. and weighed periodicallyuntil the supply of feed became exhausted in any one group, After 4 weeks the feed supply in one group became exhausted. At'this time, all chicks in each group On the fourth day of feeding, chicks in the group a which were being maintained on the feed of Lot 1, showed signs of the early symptoms of the hemorrhagic syndrome, that is, depression and fetid diarrhea. This condition prevailed until the 17th day. whendepression was no longer observed; however, diarrhea in this group was observed on the 21st day. Thereafter, chicks in this group grossly appeared normal.

The secondgroup of chicks which are m were's'acrificed by decapitationand examined at autopsy for gross manifestation of poultry hemorrhagic syndrome.

7 The degree of fungal proliferation in the feed samples, observed during the experiment, appears in the following table. The degree of fungal proliferation is designated as follows: 1 v

0, no growth aintained on. the feed of Lot 2, in which 2-chloro-5-nitropyridine was 4, profuse growth ;As indicated in the following table, the lots of feed confindings, that although taining from to 100 p.p.m of 2-chloro-5-nitropyridine showed fungal proliferation-after 14 days. No fungal growth was. observed in the samples containing 250 and 500 p.p.m. of 2-chloro-5-nitropyridine.

Table 2 Growth on Days Indicated, Days Concentration Compound (p.p.m.)

' e s 1o 14 0 1 4 4 4 i0 1 4 4 4 b0 i 1 1 1 100 0 0 0 1 mo 0 o o r It can'be conclusively stated, as a result of autopsy the 2-chloro-5-nitropyridine did not suppress fungal poliferation in the lots containing 10-100 p.p.m. of the compound, the chicks that consumed these lots showed a pronounced suppression of the hemorrhagic syndrome. It has been observed in many experiments that chicks used as controls and which re- 1 ceived so-called normal feed as it is purchased on the openmarket, at autopsy, had an average hemorrhagic score of 4 to 5. Thus, the mean scores of 5 for-50 p.p.m. level, as well as those for the 250 p.p.m. levels and above can be considered to approximate normal values. It can also be stated that the 2-chloro-5-nitropyridine in the concentrations tested had no apparent effect on gain in body weight. The results of the autopsy are set forth in the following table, again utilizing the arbitrary hemorrhagic syndrome score set forth hereinabove.

. Table 3 Concentration oi Compound (p.p.m.)

0 50 100 200 250 300 400 500 Number of chicks Autopsied 10 10 10 10 10 10 10 9 10 Total "Hemorrhagic Syndrome" Score 223 83 61 71 53 48 33 28 24 Mean Hemorrhagic .Byndrome Score 22 8 6 7 5 5 .3 3 2 EXAMPLE 4.'PRODUCTION OF THE HEMOR- RHAGIC SYNDROME UNDER FIELD CONDI- TIONS The relationship of the toxic fungal elaboration products to the hemorrhagic syndrome was confirmed by an effective production of the hemorrhagic syndrome under field conditions. by concurrently suppressing the hemorrhagic syndrome under field conditions, using the fungicidal concentration of 2-chloro-S-nitropyridine.

Production of the hemorrhagic syndrome.Normal broiler mash having a moisture content of 12.8 percent, containing no antibiotic or coccidiostate, was inoculated with 0.5 percent of dry inoculum of the mixture of toxic fungi described in Example 1. The feed was then fed to chicks maintained on woodshavings litter on a concrete floor in a building having a total area of 100 square feet. One hundred chicks, 50 males and 50 females, had free access to the feed in hoppers and water in normal watering devices located on the litter. Litter and feed subsequently scattered in the litter by the chicks, were examined periodically by steriomicroscopic means for fun- The relationship was further validated,

gal proliferation, and the festation of toxicity.

Nineteen days after initiation of the experiment, slight fungal proliferation was observed in feed scattered by the chicks in litter vnear water devices, and heavy fungal growth was observed three days later. Fungal prolifera-' tion gradually became more evident in feed scattered in other parts of the chicken house and wa feed and litter in all parts of the house by the fourthweek.

0n the 22nd day of the experiment, slight depression, diarrhea, and paleing of combs and Wattles were observed in some chicks and become more pronounced in approximately 25 percent of the flock at 4V2 weeks. Death began to occur in chicks at 4 weeks and mortality was 10 percent at 5 /2 weeks.

Gross autopsy findings were typical of those observed in field cases of poultry hemorrhagic syndrome, namely, superficial and deep hemorrhages and congestion in the musculature of the legs, thighs, and breasts; hemorrhages and congestion in the lungs, liver, spleen, kidneys, small intestine and caeca, and pronounced erosion in the proventriculus and gizzard. At 7% weeks, when the experimcnt was concluded, moderate depression and fetid diarrhea were observed in practically 100 percent of surviving chicks. Five males and five females sacrificed by decapie tation showed gross autopsy findings of severe manifestations of poultry hemorrhagic syndrome having an average hemmorrhagic syndrom "score of 30.

It was also determined that the hemorrhagic syndrome exists in two forms, namely the acute and chronic. The acute form appears after ingestion of large quantities of a highly toxic substrate, and is characterized for the'most part by a hemorrhagic diathesis and ploethora, and degenerative changes in the liver and kidneys. When small amounts of toxic feed are administered over a long period of time animals develop various stages of chronic toxicoses. The most outstanding symptom of the final stage of the chronic syndrome is pronounced depression of the haemophoeitic system. Suppression of the hemorrhagic syndrome-To conclusively prove the relationship of the toxic fungal metabolic by-products to the hemorrhagic syndrome, it is believed that a suppression of the fungal proliferation in feed and litter will concomitantly result in a suppression of the poultry hemorrhagic syndrome in chicks maintained under field conditions which would be substantially the same as those described in the foregoing production of the hemorrhagic syndrome.

To the inoculated feed were added by rubbing through chicks observed for gross mani- -mesh screen, 500 p.p.m. of 2-chloro-5-nitropyridine mixed for 15 minutes in a cement mixer and fed to chicks.

Steriomicroscopic examination of litter and feed scattered in litter by the chicks for 9 weeks did not demonstrate fungal proliferation. In addition, during this period the chicks demonstrated no evidence of poultry hemorrhagic syndrome. Five males and five female chicks were sacrificed at this time by decapitation. 'The autopsy findings resulted in showing that the chicks for all practical purposes were normal, having an actual average syndrome score of less than 1.0. This is a striking comparison to the fact, that as indicated in Tables 1 and 2, the chicks that consumed the feed with the inoculum alone demonstrated an average syndrome score of 16.

I claim: g

1. A method of so treating cereals as to prevent the formation therein, by the action of fungi in the presence of moisture of amounts of metabolic. by-products that will be toxic to animals ingesting the same, which comprises incorporating into said cereal 10-100 p.p.m. of 2-chloro-5-nitropyridine.

2. A method according to claim 1 in which the con I centration of 2-chloro-5-nitropyridine is within the range s pronounced in it capable of producing toxic levels acts in conditions of relatively high humidity, containing of metabolic by-proda concentration of Z-chIoro-S-nitropyridin, which is within the'rangc of 10-4000 p.p.m.

4. A'composition according to claim 3 in which the 8 concentration of 2-chloro-5-nitropyridine is within the range f 30-500 p.p.m.' I

Norefcrences cited.

5 A. LOUIS MONACELL, Primary Examiner.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION atent Non 3,208,852 September 28, 1965 Joseph Forgacs Column 3, line 41 for "2-CHLORO--NITROPYRIDINE" read 2-CHLORO-5-NITROPYRIDINE Signed and sealed this 19th day of April 1966a TEST W. SWIDER sting Officer Commissioner of Patents EDWARD J. BRENNER 7 

1. A METHOD OF SO TREATING CEREALS AS TO PREVENT THE FORMATION THEREIN, BY THE ACTION OF FUNGI IN THE PRESENCE OF MOISTURE OF AMOUNTS OF METABOLIC BY-PRODUCTS THAT WILL BE TOXIC TO ANIMALS INGESTING THE SAME, WHICH COMPRISES INCORPORATING INTO SAID CEREAL 10-100 P.P.M. OF 2-CHLORO-5-NITROPYRIDINE.
 3. A CEREAL, WHICH WHEN INFECTED WITH FUNGI IS NORMALLY CAPABLE OF PRODUCING TOXIC LEVELS OF METABOLIC BY-PRODUCTS IN CONDITIONS OF RELATIVELY HIGH HUMIDITY, CONTAINING A CONCENTRATION OF 2-CHLORO-5-NITROPYRIDINE, WHICH IS WITHIN THE RANGE OF 10-1000 P.P.M. 